Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/1012—Calibrating particle analysers; References therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
  • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
  • G01J3/28—Investigating the spectrum
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
  • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
  • G01J3/28—Investigating the spectrum
  • G01J2003/2866—Markers; Calibrating of scan
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/967—Standards, controls, materials, e.g. validation studies, buffer systems
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/80—Fluorescent dyes, e.g. rhodamine
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
  • Y10T436/101666—Particle count or volume standard or control [e.g., platelet count standards, etc.]

Definitions

• This invention relates generally to a method and kit for calibration of a flow cytometer or fluorescence microscope, and for performing quality control and quantitating binding proteins on samples.
• Fluorescence cytometry with flow cytometers or fluorescence microscopes is useful for studying particular cells which have been labeled due to the binding of specific labeled antibodies or other labeled proteins such as interleukins, antigens, cytokines, hormones, enzymes and filamental proteins.
• Monoclonal antibodies are used as analytical probes for the detection of cell surface antigen expression, for example, on lymphocytes, granulocytes, platelets, erythrocytes, eosinophils, basophils, and stem cells to aid in clinical diagnosis and medical treatment of a variety of immunological diseases. Changes in the levels and ratios of different types of cells are often associated with particular diseases. Fluorescent labeling of antibodies which bind to these types of cells, or of other proteins which bind to the cells, together with flow cytometry, allows detection of cell populations which occur in small numbers and quantitation of fluorescence intensity and epitope expression.
• Fluorescent intensity microbead standards together with a single antibody binding population can be used to determine the "effective F/P ratio" which is the ratio of fluorescent dye molecules to the number of antibody molecules.
• Knowledge of the effective F/P ratio allows calculation of the average number of antibodies binding to cells which is determined by dividing the total number of fluorescent dye molecules associated with a particular antibody-labeled cell, determined via instrument calibration with appropriate microbead standards, by the F/P ratio.
• Antibody binding calibration microbead standards termed SIMPLY CELLULAR ® microbeads, have been developed which directly bind antibodies and are used to construct calibration curves and determine the antibody binding capacity of labeled samples (U.S. Patent No. 4,918,004).
• SIMPLY CELLULAR ® microbeads have been developed which directly bind antibodies and are used to construct calibration curves and determine the antibody binding capacity of labeled samples (U.S. Patent No. 4,918,004).
• the types of kits and microbeads known in the art it often is inconvenient when the standards need to be labeled in a separate step, and/or the calibration line has to be plotted on graph paper.
• quality control is poorly defined in many systems due to a lack of a standardized mathematical definition of the calibration line and the performance parameters (e.g., sensitivity, linearity, dynamic range).
• a method and t for calibrating a flow cytometer or fluorescence microscope and for quantitating binding antibodies, or other proteins comprising providing populations of microbeads which are in equilibrium with a saturating amount of fluorescently-labeled protein, such as a fluorescently-labeled antibody.
• the microbeads preferably have an incorporated fluorescent dye which has a strong signal in a selected channel of a flow cytometer, but no signal in other channels.
• the kit includes a suspension containing protein-binding microbeads in equilibrium with the specific proteins which bind to the microbeads; and preferably, a software product containing a program for determination of selected performance parameters for quality control prior to dete ⁇ riining the protein-binding capacity of samples. Additional information is preferably provided on the calibrated binding capacities of the various microbead populations.
• Figure 1 is a bivariant forward v. side angle light scatter histogram of polymeric microbeads mixed with human leucocytes indicating that there is overlap of the microbead and cell signals (described in Example IV).
• the cell signals the smaller lower left signals are the lymphocytes, the small central signals are the monocytes, and the larger signals overlapping the microbead signal are the granulocytes.
• Figure 2 is a bivariant forward angle light scatter vs. FL3 histogram of polymeric microbeads mixed with human leucocytes indicating that there is complete resolution between the microbeads and cell signals (described in Example VII).
• the gated cells are granulocytes, with the monocyte signals, and then the lymphocyte signals positioned below the granulocyte signal.
• Figure 3a is a FL1 histogram from gated microbeads (3a) in Figure 2.
• Figure 3b is a FL1 histogram from gated cells (3b) in Figure 2.
• Figure 4 is an example of a calibration plot according to the invention showing antibody binding capacity vs. histogram channel in Example VHQ. The plot shows data points (+) for the microbead populations, best fit ( ⁇ ), and data point for the blank microbeads (O).
• the present invention provides a method for calibrating a flow cytometer for use with fluorescently-labeled proteins which include antibodies, interleukins, antigens, cytokines, hormones, enzymes, and filamental proteins.
• the invention comprises a plurality of microbead populations which have a component on their surface which will bind different specific amounts of the selected proteins (called specific populations), including a microbead population which has no specific binding capacity for the selected protein. This plurality of microbead populations is suspended in a solution of excess (e.g., about 3-fold) selected protein to maintain equilibrium binding.
• the microbead populations preferably contain a fluorochrome which is easily measured by one of the fluorescence detectors of the flow cytometer, but is undetectable by the other fluorescence detectors.
• the microbeads contain a far red fluorescence detectable by the FL3 detector, but which has no fluorescence signal in the FL1 or FL2 detectors.
• having the populations of microbeads "in equilibrium" with the saturating amount of fluorescently-labeled protein means that these components have been together in the mixture thereof for at least about 24 hours and preferably for at least a week prior to being used. In equilibrium, while there generally is not 100% saturation of the binding sites, one expects practically to have over about 90% saturation, and most likely over 95% saturation, when the mixture is at equilibrium, as in the invention herein.
• the suspension of microbeads is washed prior to obtaining the calibration plot.
• the microbeads are added, without prior washing, to sample cells such that the excess fluorescent protein labels the sample cells, forming a mixture containing fluorescently-labeled microbeads and fluorescently-labeled sample cells.
• the mixture is incubated, lysed, and washed using standard procedures (for example, resuspend in phosphate-buffered saline, spin down in a centrifuge at about 2000G, and repeat resuspension and centrifugation at least one more time).
• the washed mixture is analyzed by gating on the microbeads in the mixture to determine representative peak channels, and using the representative peak channels and previously provided binding capacities of the microbeads to obtain a calibration plot, and to obtain performance parameters.
• the fluorescently-labeled sample cells can then be gated with the flow cytometer or fluorescence microscope (assuming that the cells and microbeads are separately identifiable with a flow cytometer or fluorescence microscope) to determine the protein-binding capacity of the sample cells.
• Gating refers to the practice of taking a selected population, for example, based on light scatter, and doing analysis only on that population, so that the resulting analysis is for that specific population only and is not made inaccurate by analysis of a mixed population.
• Gating preferably includes obtaining a dot plot of forward angle vs. the selected fluorescence channel for the fluorescent dye that the beads are stained with, drawing a box around groups of dots on the dot plot of the selected resolved populations of microbeads or sample cells, and obtaining a fluorescent histogram of each group using standard flow cytometry techniques.
• first fluorescent dye has a strong signal in a selected channel of a flow cytometer, but no signal in other fluorescence channels where the fluorescently-labeled protein has a signal.
• a useful dye for this procedure is oxazine I (Polyscience, Inc., Warrenton, PA).
• fluorescently-labeled protein is labeled with a second, different dye, for example, fluorescein isothiocyanate or phycoerythrin.
• This refinement of the method and kit of the invention allows resolution of the microbeads from the cells by gating, because the microbeads labeled with the first fluorescent dye can be pulled out and analyzed separately from the cells without contaminating one population with the other.
• the method and kit of the invention may be used with any type of cell sample as is known in the art, for example, lymphocytes, granulocytes, platelets, erythrocytes, eosinophils, basophils, and stem cells.
• the method and kit of the invention include a software product which operates on a computer and contains information on the fluorescence intensity of each population of microbeads, and operates with the computer to control calculation of calibration and fluorescence information tailored to the microbead populations, for determination of selected performance parameters for quality control.
• the method and kit of the invention may be used for increasing accuracy, precision and reliability of studies of binding quantitation of protein-labeled samples using a flow cytometer or fluorescent microscope.
• any type of microbeads which are uniform in size and capable of uniformly binding the antibodies or other proteins which are to be used, may be employed.
• Preferred microbeads include those disclosed, for example, in U.S. Patent No. 4,767,206, but microbeads having substantially different compositions than these may also be used.
• the features and advantages of the present invention will be more clearly understood by reference to the following examples, which are not to be construed as timiting the invention.
• the first five examples describe experiments illustrating problems with prior calibration and quantitation methodologies, and the remaining examples relate to aspects of the invention herein.
• microbead having the characteristics of high uniformity of size and distribution of functionally groups may be used in the method and kit of the invention.
• One method of microbead preparation is as follows: suspend a highly uniform population of microbeads (i.e., having a size of 7-10 ⁇ diameter and a coefficient of variation in diameter of about 2% or less within a population, which contain free carboxyl groups on their surface in 0.1 M HEPES at pH 4.5, and activated with a water soluble carbodiimide (1 ethyl-3-(3-dimethylaminopropyl) carbodiimide). Wash twice with 0.1 M HEPES at pH 4.5, then with 0.1 M HEPES at pH 7.0.
• binding protein e.g., goat anti-mouse IgG antibody
• PBS phosphate-buffered saline, pH 7.2
• Antibody-binding capacities of four different microbead populations made according to Example I and being Fc specific are dete ⁇ nined for three different sources of two CD antigens.
• the same four microbead populations (1-4) were used for each of the four types of labeled antibodies (CD4-FTTC, CD4-PE, CD8-FTTC, and CD8-PE).
• the different antibodies compared in each group are specific for the same type of cells (CD4 or CD8) and are bound to the same fluorochrome.
• Binding capacities are determined by direct relative linear channel proportionality (where the scale is directly proportional to the fluorescence intensity) with pooled normal peripheral lymphocytes using each respective antibody against levels of 50,000 for the CD4 antigen and 180,000 for the CD8 antigen. These levels are specified in the literature (e.g., Poncelet, P. et al., Methods of Immunol. Anal., eds. Y. Masseyeff et al., 1993, 3:388-417) as being the binding capacity of these lymphocytes. Results are shown in Table 1.
• the average binding capacities of the four antibodies are shown in Table 2 below along with their percent coefficient of determination. This type of determination is preferably used to determine the assigned ABC of particular microbead populations for use in the method and kit of the invention herein. These results show the wide difference in binding capacities for the same microbead populations. Table 2.
• CD3 clone pan-lymphocyte marker
• FITC or PE Sigma Immunochemicals
• Microbeads prepared as in Example I are exposed at room temperature for different amounts of time to the same antibody at either 10 ⁇ l or 20 ⁇ l antibody per 100,000 microbeads. These concentrations are at or near saturation of the binding capacity of the microbeads.
• the antibody used in this example is anti-CD4 obtained from Becton Dickinson Immunocytometry Systems, conjugated with fluorochrome phycoerythrin.
• the flow cytometer used for this study is a FACScan (Becton Dickinson Immunocytometry Systems). Results are shown in Table 4. Similar results are obtained with other types of microbeads that are uniform in size and are capable of uniformly binding the selected antibody or protein.
• Table 5 shows the intensity channel stability (peak channels) on a 1024 scale from mouse IgG binding microbeads, using a different lot of the same four populations of microbeads discussed above, which have been left to form an equilibrium with CD4-FITC antibody (Sigma Immunochemicals) for the specified number of hours prior to washing two times and analyzing with a FACScan Flow Cytometer. By the 96 and 385 hour sampling, an equilibrium had been reached.
• FL3 channels show a signal brighter than the autofluorescence of the cells, but show no increase of intensity over unlabeled microbeads in the FL1 and FL2 channels, as shown in Figure 2.
• Separate gating of the microbeads and cells is then accomplished using the FL3 vs. forward angle scatter bi-variant histogram as shown in Figures 3a and 3b.
• EXAMPLE VHT Establishment of a Calibration Plot, Determination of Performance Parameters and Quantitation of Binding Capacitv using a Computer Program
• Pre-dyed (Oxazine I) goat anti-mouse binding microbeads in equilibrium with excess CD4-FTTC antibody from Coulter Corporation are added to an equal volume (100 ⁇ l) human peripheral whole blood and allowed to incubate at room temperature for 30 minutes.
• the blood in the microbead mixture is then lysed with BDIS Lysing Solution for ten minutes and washed twice with PBS containing 0.5% paraformaldehyde.
• the sample is run on a FACScan flow cytometer (BDIS) and 20,000 events collected (an "event" is anything that triggers the instrument such as a microbead or cell).
• the listmode file is analyzed by first gating the microbeads in the forward vs.
• Goat anti-mouse IgG binding microbeads in equilibrium with excess unlabeled CD8 antibody are added to human peripheral whole blood, incubated for 30 minutes and washed three times with PBS containing 0.2% bovine serum albumin (BSA). These are then labeled with a fluorescent ligand, such as goat anti-mouse conjugated to PE secondary antibody, and the blood lysed with BDIS Lysing Solution for ten minutes. After washing three times with PBS, the sample is run on a FACScan flow cytometer, and the listmode data analyzed by gating on the microbeads and cells respectively to determine their peak channels. These peak channels are analyzed with the QUICKCAL v.
• BSA bovine serum albumin
• microbeads of Example I Two sets of calibrated binding microbeads (four populations per set) were prepared of different sizes, with the microbeads of a first set of microbeads having a diameter of 7 ⁇ and the microbeads of the other set having a diameter of 10 ⁇ .
• the microbeads of these two sets had specific CD4 or CD8 antigens, respectively, conjugated to their surface.
• microbead concentration of 2 x 10 6 per ml 100 ⁇ l of whole blood was added.
• Fifteen microliters of anti-CD4-FTTC and anti-CD8-PE (Becton Dickinson) were added to the mixture and allowed to reach equilibrium overnight according to the invention herein.
• the mixture was then lysed with FACS Lyse (Becton Dickinson) and washed twice with
• CD4-FTTC mouse monoclonal antibody (Sigma Biosciences, St. Louis, MO) at a concentration of 50 ng/ml, plus one ml PBS, was analyzed in a Shimatzu Spectrofluorometer and found to have a fluorescence reading of 15.74.
• the binding capacity of other proteins can be determined by the same method, and measurable labels other than fluorescent labels may be used, such as radiotracers, so long as they can be correlated with the concentration of the protein.

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• 1995
  • 1995-12-27 US US08/579,186 patent/US5837547A/en not_active Expired - Lifetime
• 1996
  • 1996-12-24 DE DE69633344T patent/DE69633344T2/de not_active Expired - Lifetime
  • 1996-12-24 AU AU15671/97A patent/AU1567197A/en not_active Abandoned
  • 1996-12-24 EP EP96945415A patent/EP0870195B1/de not_active Expired - Lifetime
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